Using retroviral insertional mutagenesis, we identified SENP1, a protease regulating the dynamic process of post-translational modification by the ubiquitin-like protein SUMO, as a developmentally essential gene. SUMO modification of various proteins, by a process similar to but distinct from ubiquitination, regulates a number of critical biological processes. SENP1 is one of several higher eukaryotic genes related to yeast Ulp1, which encodes a cysteine protease that can cleave SUMO from modified proteins as well as carry out the maturation of the SUMO precursor. Insertional mutation of SENP1 causes a dramatic increase in the steady state levels of the sumoylated forms of a number of proteins. However, only the level of SUMO-1 conjugation increases; levels of the related modifiers SUMO-2,-3 are unaffected. In addition, mutant cells show a decrease in free SUMO-1 and an accumulation of the unprocessed form of SUMO-1. These results have proven a role for SENP1 in both deconjugation and maturation of SUMO-1, as found for yeast Ulp1. The physiological consequences of loss of SENP1 function during development are first apparent after midgestation in the placenta. These defects appear incompatible with normal placental function and embryonic viability. Current work is focused on the sumoylated proteins that show increased levels in mutants. We have isolated primary mouse embryonic fibroblasts (MEFs) from mutants and wild types and introduced an epitope tagged form of SUMO-1. Preliminary results show that this form of SUMO-1 can be efficiently conjugated to proteins and that these sumoylated proteins can be specifically immunoprecipitated. The significant increase in steady state levels of sumoylated proteins in the mutant should now permit proteomic approaches to determine their identities and allow an assessment of their potential roles in the mutant phenotype. As a second approach to identify SENP1 desumoylation substrates in the developing embryo, we are generating transgenic mice carrying a conditionally active dominant negative form of the SENP1 gene. Results in tissue culture indicate that the catalytically inactive enzyme can bind substrates and form stable complexes, allowing the sumoylated proteins to be co-immunoprecipitated.